Rf2a and rf2b transcription factors

ABSTRACT

A method of activating the rice tungro bacilliform virus (RTBV) promoter in vivo is disclosed. The RTBV promoter is activated by exposure to at least one protein selected from the group consisting of Rf2a and Rf2b.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application is a §371 application of PCT/US01/50748, filedOct. 23, 2001, which claims priority to U.S. provisional patentapplication Ser. No. 60/242,908, filed Oct. 24, 2000. Each of theabove-identified application is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work of this invention is supported in part by a grant from theDepartment of Energy. The United States Government has certain rights tothis invention.

FIELD OF INVENTION

The present invention relates to the field of plant molecular biology.This invention concerns methods of regulating gene expression intransgenic plants. In particular, this invention discloses isolated andpurified proteins which act as transcription factors capable ofactivating promoters to enhance gene expression.

DESCRIPTION OF RELATED ART

Bhattacharya—Pakrasi, et al., in. “Specificity of a promoter from a ricetungro bacilliform virus for expression in phloem tissues,” The PlantJournal, 4(1):71-79 (1993) identified the promoter sequence in the ricetungro bacilliform virus (RTBV) genome. The article also reported thatgene expression under the control of tie RTBV promoter occurredprimarily in vascular bundles, particularly phloem and phloem associatedcells in leaves.

Yin and Beachy, in “The regulatory regions of the rice tungrobacilliform virus promoter and interacting nuclear factors in rice(Oryza sativa L.),” The Plant Journal, 7(6) 969-980 (1995), describedthe E fragment (−164 to +45 in relation to the transcription start site)within the RTBV promoter which is sufficient to cause tissue-specificgene expression. The article also disclosed a critical cis element, BoxII (−53 to −39), as a binding site for rice nuclear factor 2 (RNFG2)which is essential for promoter activity.

The same authors also identified other cis elements. Yin, et al.,“Promoter elements required for phloem-specific give expression from theRTBV promoter in rice,” The Plant Journal: 12(5): 1179-1188 (1997).These other cis elements are the ASL Box (−98 to −79) and a GATA motif(−143 to −135). Together, these cis elements (−164 to −32) conferredphloem-specific reporter gene expression. These regions act additivelyto direct tissue-specific expression.

Yin, et al., in “Rf2a, a bZIP transcriptional activator of thephloem-specific rice tungro bacilliform virus promoter, functions invascular development,” The EMBO Journal, 16, (17): 5247-5259 (1997),identifies a 1.8 kb transcription factor protein composed of 368 aminoacids designated Rf2a. Rf2a binds to the Box II element and stimulatesBox II—dependent transcription in vitro. When Rf2a protein accumulationis suppressed in transgenic plants, morphological changes—includingstunted plants, twisted leaves which contain small disorganized vascularbundles, enlarged sclerenchyma and large intracellular airspores—appeared. The article teaches that the RTBV promoter interactswith a host transcription factor critical for leaf tissuedifferentiation and vascular development.

Beachy, in U.S. Pat. No. 5,824,857 entitled “Plant Promoter,” describesthe promoter from the rice tungro bacilliform virus (RTBV). Thedisclosure teaches that the RTBV promoter causes preferential expressionin plant vascular tissue. The patent also teaches that the RTBV promotercan be used to drive expression in most plants, whether monocotyledonousor dicotyledonous, and is particularly suited to rice. The disclosurealso teaches the transformation of plants by inserting the codingsequence of the promoter and a heterologous gene of interest to obtaintransgenic plants which express the gene of interest in vascular tissue.

In plant biotechnology, selection of suitable promoters is crucial toobtaining expression of the gene of interest. Tissue specific promotersdrive gene expression in a certain specific tissue or in severalspecific tissues of the plant. Constitutive promoters, on the otherhand, drive gene expression in most tissues of a plant. Whileconstitutive promoters are more widely used in the industry, tissuespecific promoters are useful, or even necessary for some applications.A method of activating a tissue specific promoter to drive constitutegene expression thereby giving a greater degree of control overexpression of the transgenes would be a valuable and useful tool to theindustry.

Regulation of gene transcription is achieved by the activity of multipletranscription factors that bind to regulatory elements, many which areupstream of the promoters and alter basal rates of transcriptioninitiation and/or elongation (1, 2). To understand the mechanisms oftissue-specific and constitutive gene expression in plants, a number ofplant promoters and transcription factors have been studied in recentyears (3-16). It was shown that constitutive promoters, such as theCauliflower Mosaic Virus (CaMV) 35S promoter (17) and the promoter fromCassava Vein Mosaic Virus (CVMV) (14) have modular organizations, eachof the multiple cis elements within the promoters confer cell typespecific gene expression. These elements apparently interact in anadditive and/or synergistic manner to control the gene expressionpattern in all plant tissues. Similarly, tissue-specific promoterscontain multiple elements that contribute to promoter activity in bothpositive and negative ways (4-6, 8, 12, 18). All these cis elementsprovide DNA targets for binding by transcription factors, thecombinations of which contribute to regulation of gene expressioncontrolled by the promoter.

The rice tungro bacilliform badnavirus (RTBV) promoter and thetranscription factors that interact with this promoter serve as a modelsystem to study plant tissue specific gene expression. RTBV is a plantpararetrovirus with a circular double-stranded DNA genome from which thepregenomic transcript is produced (19, 20). The virus, which replicatessolely in phloem tissues, has a single promoter that is active intransfected protoplasts and is phloem specific in transgenic rice plants(7, 10, 21, 22).

Within the fragment E of the promoter (nucleotides −164 to +45),multiple cis elements were identified as being required for phloemspecific gene expression and promoter activation (7, 10, 15, 23). A bZIPtype transcription factor isolated from rice plants—Rf2a—binds to BoxII, a crucial cis element of the RTBV promoter. It was subsequentlyshown that Rf2a activates transcription from this promoter in a rice invitro transcription system (10, 11, 15). Moreover, studies in transgenicrice plants suggested that Rf2a is involved in the development ofvascular tissues (11).

With the advancement of plant biotechnology comes the need for strongactivation systems to regulate gene expression in transgenic plants.Non-plant transcription factors present a solution, however, given thedifferences between plants, mammals, Drosophila and yeast (24), theefficiency of such non-plant transcription factors may be negativelyaffected. It is important to develop a plant-based transcriptionalregulatory system in the whole plant level. Accordingly, this inventionteaches a plant-based transcriptional regulatory system based on theinteraction between the transcription factors Rf2a and Rf2b and the RTBVpromoter.

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to provide transcription factors whichactivate the RTBV promoter such that the promoter causes constitutiveexpression of the genes under its control.

This invention identifies the transcription factors Rf2a and Rf2b. Theinvention also teaches the function of the RTBV promoter in transgenictobacco plants and the interaction between the transcription factors andthe promoter. Methods of regulating gene expression are also provided.

This invention describes Rf2a, a bZIP protein that binds to the Box IIcis element of the RTBV promoter, and Rf2b, the putative partner forRf2a. Rf2b is capable of forming a heterodimer with Rf2a. The bZIPdomain of Rf2b shares 72% nucleiotide identity with Rf2a. At the aminoacid level, the two proteins share 83% identity. Rf2b has two putativefunctional domains: an acidic domain at the N-terminus and a prolinerich domain at the C-terminus. Rf2b can bind to Box II and the mutantBox IIml (where the CCCC sequence is modified to GCGC).

This invention also provides a method of activating the RTBV promoter invivo by exposing it to transcription factors Rf2a and/or Rf2b. As usedherein, the term “activate” or “activating” means to regulate such thatenhanced or constitutive expression of those genes which are under thecontrol of the promoter results. The method of “exposing” the RTBVpromoter comprises cloning the RTBV promoter coding sequence into aplasmid, cloning the coding sequence for Rf2a or Rf2b into a plasmidexpression vector containing a CaMV 35S promoter and a Nos terminator,and cloning the plasmid expression vector containing the CaMV 35Spromoter and the Nos terminator and either Rf2a or Rf2b sequence intothe plasmid. Target plant cells would then be transformed usingtechniques known to those skilled in the art. The transgenic plant cellswould then be allowed to grow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A: Schematic representation of the RTBV full-length promoter(FL) and of the E fragment (E) of the promoter. The cis elements GATA,ASL, Box II and Box I of E (described by Yin et al., Plant J. 1997) areindicated. Box II, the cis element to which Rf2a binds, is indicated inblack.

B. Schematic representations of the Rf2a transcription factor and of the3Δ mutant. The potential activation domains are indicated as P forproline rich; A for acidic region; and Q for glutamine rich region. Thedimerization and DNA binding domain is indicated as bZIP (Yin et al.,Embo J. 1997).

C. Diagram of the constructs used for Agrobacterium mediatedtransformation of tobacco (N tabacum). Uid A gene (GUS encoding gene) isdriven either by the Full Length rice tungro bacilliform virus promoter(FL) or E fragment (−164 to +45). The genes encoding the ricetranscription factor Rf2a and the 3Δ mutant are driven by the 35Spromoter of CaMV.

FIG. 2: Histochemical localization of GUS in tissue from transgenictobacco plants. A, C, E, L, K: plants containing either PE:GUS orPFL:GUS genes. B, D, F, H, J, L: plants containing eitherPE::GUS(+)P35S::Rf2a or PFL::GUS(+)P35S::Rf2a. The results shown arerepresentative of those observed in the 15 independent transgenic plantsdeveloped for each gene construct. Results with PE::GUS and PFL::GUSconstructs were identical, and the figure is compiled from both sets oftransgenic plants. GUS activity is indicated in transgenic tissue by anindigo dye precipitate after staining with X-Gluc. A, B: tobaccoseedlings; C, D; juvenile leaves; E, F: roots; G, H: leaf sectionsshowing vascular tissues of the midrib and leaf lamina; 1,3: vasculartissue of the leaf midrib; K, L: cross section of lamina. (c, cotyledon;cr, cortex; e, epidermis; ex, external phloem; g, guard cell; ip,internal phloem; 1, leaf, m, mesophyll; p, phloem; pm, palisademesophyll; py, parenchyma; rt, root tip-rv, root vein; sm, spongymesophyll; t, trichome; vb, vascular bundle; x, xylem.)

FIG. 3: GUS activity in extracts of leaves from Ro transgenic tobaccoplants carrying the PE::GUS gene, PE::GUS(+)P35S:Rf2a, PFL::GUS orPFL:GUS (+)P35S::Rf2a. The amount of GUS (pmoles 4−MU min⁻¹ mg⁻¹protein) is indicated. The mean level of GUS activity for each constructis indicated by the thick, horizontal bar.

FIG. 4: Correlation between GUS activity and the amount of Rf2adetermined by ELISA for transgenic T₀ plant lines that carryPE::GUS(+)P35S:Rf2a or PFL:GUS (+)P35S::Rf2a.

FIG. 5: Electrophoretic mobility shift assay (EMSA) of Rf2a and the 3Δmutant of Rf2a. Oligonucleotides containing the BoxIIml sequences (Yinet al., 1997a) were used as ³²P-labeled probe or an unlabeled competitorin an EMSA with 500 ng of E. coli protein containing 3Δ protein or Rf2a.Unbound probe is located near the bottom of the gel. The band with thegreatest mobility in lanes 1, 2, 4, and 5 indicated by x is presumed toresult from binding with an uncharacterized protein that co-purifieswith the target protein. Lane 1, protein prepared from E. coli withoutinduction. Lane 2, reaction with extracts of E. coli that produce Rf2a.Lane 3, reaction with equimolar amounts of Rf2a and 3Δ plus 80× foldmolar excess of unlabeled competitor probe relative to the labeledprobe. Lane 4, reaction with equimolar amounts of Rf2a and 3Δ. Lane 5,reaction with 3Δ.

FIG. 6: Phenotypes of plants that contain the gene encoding the 3Δmutant of Rf2a A, C, segregation of the stunting phenotype with thetransgene. A: the plants on the left contain the transgene and grew moreslowly than the plant that lacked the transgene due to segregation inthe T₁ generation (right). C: size of roots in plants with the 3Δ gene(plants on the right) compared with non-transgenic T₁ progeny (left). B:intermediate phenotype, showing shoot elongation, curvature of leavesand decreased apical dominance. D, E: close up of leaves showingdownward curvature. F: abnormal plant showing the phenotype of severestunting with thick leaf lamina.

FIG. 7: A schematic diagram of the domain structures of Rf2a and Rf2b,and an alignment of amino acid sequences in the bZIP domain of Rf2a andRf2b with other known bZIP proteins (piece of SEQ ID NO: 2, SEQ ID NO:7, piece SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11 and SEQ ID NO: 12, respectively, in order of appearance, from topto bottom).

DETAILED DESCRIPTION OF THE INVENTION

Phloem specific expression of the RTBV promoter. RTBV is known toaccumulate in phloem tissues in infected rice plants. Similarly, thefull length RTBV promoter (FL) and the E fragment of the promoter areexpressed only in phloem tissues in transgenic rice plants (7, 10, 22).Within the E fragment, there are several cis DNA sequence elements thatare conserved among vascular tissues (10). Box II is a cis element thatis responsible for the basal behavior of the promoter. Box II shareshomology with cis elements from other phloem and xylem specificpromoters (10). Data from 5′ deletion analyses showed that as long asBox II cis element is retained, the promoter is expressed in phloemtissues, although activity of the promoter is much lower in the absenceof Box II than when it is present. It is known that some plant promotersretain specific expression patterns in different plant species (20-31)while others do not (32,33).

Rf2a can activate the promoter in cell types other than phloem cells. Aswe reported previously, the RTBV promoter contains several important ciselements, such as Box II, that contribute to expression of the promoterin vascular tissues. Data from 5′ deletion analyses showed that as longas the Box II cis element is retained, the promoter is expressed inphloem tissues, although activity of the promoter is much lower in theabsence of Box II than when it was present (10). He et al (15) reportedthat when the element, which contains Box II, was deleted or mutated inthe context of the E fragment, the promoter activity dropped to basallevels.

To better understand the interactions between Rf2a and the RTBVpromoter, the following plasmids were constructed: pGA-FL::GUS, whichcontains the full length RTBV promoter and the uidA reporter gene, uidA;pGA-E::GUS, which contains the E fragment and the uidA reporter gene;and pET-Rf2a, which contains the coding sequence for Rf2a. To study theeffects of co-expression of Rf2a and the RTBV promoter, co-expressionplasmids were constructed whereby the fusion gene P35S::Rf2a (whichcontains the CaMV35S promoter, the coding sequence of Rf2a and a Nosterminator) was cloned into pGA-FL::GUS and pGAE::GUS. The resultantco-expression plasmids, each containing the Box II cis element, arepGA-FL::GUS/P35S::Rf2a and pGA-E::GUS/P35S::Rf2a.

When plant cells were transformed with the co-expression plasmid, thepattern of GUS activity in transgenic plants was constitutive ratherthan phloem specific. This is consistent with the known pattern ofexpression of the 35S promoter (17) and indicates that Rf2a is involvedin regulating expression of the RTBV promoter.

Many factors can influence the activity of transcription factors incontrolling expression of the promoter, including affinity of binding oftranscription factor with the DNA element (35), the subcellulardistribution of factors, post-transcriptional modifications of thefactor (36), and synergistic interactions with other proteins (37-41).Furthermore, it is common to construct promoters by combining multipleupstream activation sequences and by replicating activation domains onDNA binding proteins. In the activation system we described here, asingle copy of Box II together with Rf2a was sufficient to increaseexpression of the RTBV promoter. In addition, the level of expressionwas positively correlated with the level of Rf2a. Other reports indicatethat expression of P-35S::GUS fusion genes in taansgenic plants rangefrom 321 U (IU is equivalent to pmol 4-MU min⁻¹ mg⁻¹) to 113,000 U (17,27, 42, 43). In our studies transgenic tobacco plants had GUS activitybetween 4,000 U and 18,000 U when P-FL::GUS or P-E::GUS were activatedby expression of the P35S::Rf2a enhancer gene, representing a 2 to 20fold activation of the reporter gene.

Although we do not yet know the basis for the strong activation of theFL and E promoters by Rf2a, there are several notable characteristics ofthis system. First, Rf2a binds to a cis element (Box II) that is locatedvery close to the TATA box (within approximately 7 nt). It is not yetknown if proximity of the cis element to the TATA sequence is importantfor the activity of Rf2a in expressing this promoter. Second, theresults of in vitro studies indicate that there is a direct physicalinteraction between TATA Binding Protein (TBP), and Rf2a (Q. Zhu et al.,in preparation). Third, Rf2a has three putative activation domains:acidic, glutamine rich, and proline rich domains (11), any or all ofwhich may be important for the activity of Rf2a. We propose thatproximity of the cis element and the unique features of Rf2a contributeto the strong activation of the promoter by Rf2a.

In transgenic plants in which the reporter genes are activated by thegene P35S::Rf2a, it is most likely that promoter activation is due toformation of homodimers of Rf2a. However, the activation byheterodimerization of Rf2a with homologous factors in tobacco cannot becompletely excluded. The observation that E fragment and FL promotersare expressed in phloem tissues in tobacco plants suggests that thereis/are Rf2a-like transcription factor(s) in tobacco.

A dominant negative mutant of Rf2a affects plant development. In riceplants, Rf2a acts as a transcription activator and its biologicalfunction is linked to the development of the vascular system: thisconclusion was based upon the results of experiments with transgenicrice plants in which the levels of Rf2a were reduced by an anti-senseapproach (11). An alternative approach to determine gene function is touse directed mutagenesis of Rf2a and determine the effects of mutationson the function of genes that require the activity of Rf2a and proteinsthat form heterodimers with Rf2a. Mutants of bZIP transcription factorshave been used in such studies and found to act as dominant negativemutants (44-46). Here, the mutant 3Δ, which contains only the DNAbinding domain and the leucine zipper region, was created to test thebiological function of Rf2a-like transcription factors in tobacco, andthe effect of 3Δ on expression of the RTBV promoter. This study, showedthat the 3Δ mutant is capable of forming homodimers. The 3Δ mutant alsoformed heterodimers with Rf2a. Both dimers bind to Box II in vitro.

The invention is exemplified by the following non-limiting example.

EXAMPLE 1

Expression pattern of the RTBV promoter in transgenic tobacco plants.The RTBV full length (FL) promoter containing nucleotides −731 to +45 ofthe RTBV genome, is expressed exclusively in phloem tissues intransgenic rice plants (7, 10, 22). A fragment of the RTBV promoter,termed the E fragment and comprising nucleotides −164 to +45, is alsoexpressed exclusively in phloem tissues (7). Within the E fragment fourcis sequence elements that contribute to phloem specific gene expressionhave been described, including the GATA motif, ASL (AS-1 like) element,Box II and Box I sequence elements (as shown in FIG. 1A) (10). Since thehost range of RTBV is limited to rice, we wished to determine whetherthe RTBV promoter is functional and maintains tissue specificity intransgenic tobacco plants. The RTBV promoter FL and the E fragment wereligated with the uid A coding sequence to produce the chimeric genesP-FL::GUS and P-E::GUS. The genes were ligated to a T₁, plasmid vector(as shown in FIG. 1A) and introduced into tobacco throughAgrobacterium-mediated transformation. 17 and 23 independent transgenictobacco plants were developed for the P-FL::GUS and P-E::GUS constructs,respectively.

A detailed histochemical analysis of GUS expression patterns intaansgenic tobacco plants showed that expression of P-E::GUS andP-FL::GUS were essentially the same (as shown in FIG. 2). In leaves oftransgenic plants with P-E::GUS, strong GUS activity was observed invascular tissues (as shown in FIG. 2A, C) although in very young leavesthere was a low amount of GUS activity in mesophyll cells (as shown inFIG. 2A). Cross sections through the midrib of more mature leaves showedGUS activity only in phloem cells (as shown in FIG. 2 G). These resultsare similar to those reported from studies of transgenic rice plants(10).

Modulation of phloem specific expression of the RTBV promoter by Rf2a.Rf2a, was isolated by virtue of its interaction with Box II DNA (10); acDNA encoding Rf2a was subsequently isolated and characterized. Rf2a isa bZIP protein with three potential functional domains: a proline-richdomain, an acidic domain and a glutamine-rich domain (as shown in FIG.1A) (11). A mutant of Rf2a that lacks these three putative functionaldomains was constructed and is referred to as “3Δ” (as shown in FIG. 1A,B). The ³A protein contains the DNA binding and leucine-zipper domains(bZIP) of Rf2a, including the putative nuclear localization signal. Todetermine the regulatory functions of Rf2a and ³A mutant on expressionof the FL and E fragment promoters in transgenic tobacco plants, thefollowing T₁, binary plasmids were constructed: PGA-E::GUS/P-35S::Rf:2a,pGA-E::GUS/P-35S::3Δ, pGA-FL::GUS/P-35S::RF2A, pGA-FL::GUS/P-35S::3Δ (asshown in FIG. 1B). At least 15 independent transgenic tobacco lines witheach construct were developed through Agrobacterium-mediatedtransformation. The integration of the full-length T-DNA in transgenicplants was confirmed by Southern blot hybridization analysis (data notshown).

Histochemical analysis of GUS activity in the TI progeny showed thatexpression of the reporter genes was substantially altered byco-expression of Rf2a. In the plants that contained eitherP-E::GUS/P-35S::Rf2a or P-FL::GUS/P-35S::Rf2a, GUS activity was detectedthroughout the cotyledonary leaves (as shown in FIG. 2B) and true leaves(as shown in FIG. 2C). Cross sections through the leaf midrib of theseplants showed very strong GUS activity in phloem cells, the epidermisand trichomes (Compare G and H in FIG. 2). The palisade and spongymesophyll cells of the leaf lamina also exhibited intense staining(compare FIGS. 2L and K) as did parenchyma cells of the midrib (compareI and J in FIG. 2). Guard cells showed strong GUS activity when theFL::GUS gene was co-transformed with P-35S::R-F2a (not shown). In theroot tissues of transgenic plants that contained P-3::GUS/P-35S::Rf2a orP-FL::GUS/P35S::Rf2a, there was a high level GUS activity throughout thecortex (as shown in FIG. 2F), and very strong levels of GUS activity inroot tips. In contrast, root tissues of transgenic plants that weretransformed only with reporter genes revealed GUS activity only in thevascular cylinder (as shown in FIG. 2E).

GUS activity in the leaves of T₀ transgenic tobacco plants wasquantified using MUG in a fluorescence assay. As anticipated, there werevariations in the amount of GUS activity among transgenic lines for eachconstruct. The GUS activity of transgenic plants with P-FL::GUS washigher than the activity in plants with P-E::GUS genes (as shown in FIG.3) When Rf2a was co-expressed with either P-E::GUS or PFL::GUS, the GUSactivity was increased by 2 to 20 fold compared with plants that lackedRf2a (as shown in FIG. 3). The increase of GUS activity is in agreementwith the observation that the pattern of GUS activity was constitutivewhen Rf2a was co-expressed with the reporter gene (as shown in FIG. 2).

To establish a correlation between the GUS activity and the amount ofRf2a, ELISA reactions were carried out to quantitatively measure theamount of Rf2a in individual transgenic plants. Soluble protein samplesof To plants were triturated and aliquots of soluble proteins weretested in quantitative ELISA reactions using anti-Rf2a antibodies.Aliquots of the same set of extracts were also used in quantitativeanalyses of GUS activity. As shown in FIG. 4, the results of theseassays showed a positive correlation between the amount of Rf2a in thesamples and GUS activity.

DNA binding and heterodimerization of Rf2a and 3Δ. The gene encoding themutant protein 3Δ, which lacks the three putative regulatory domains ofRf2a, was constructed to determine if it can restrict expression of theRTBV promoter. It has been reported that sequences outside of theleucine zipper region of such proteins can contribute to dimerstabilization (28). In these cases deletions of other (putative) domainscan affect the ability of the protein to form homo—or heterodimers, andto bind DNA. To test the DNA binding ability of mutant protein 3Δ, EMSAswere carried out (as shown in FIG. 5). Rf2a and 3Δ can each bind32P-labeled BoxIIml, presumably by forming homodimers (lanes 2 and 5).When both proteins are added, a band with mobility intermediate with themobilities of the probe to which Rf2a and 3Δ homodimers are bound wasobserved. This band is presumed to correspond to the binding of theheterodimer Rf²a/3Δ with the probe (lane 4). All of these complexes canbe competed by 80× molar excess of unlabeled cold probe (lane 3).

Morphological changes of transgenic plants with 3Δ. A gene encoding ³Aprotein was introduced along with the reporter gene into tobacco (FIG.1B), and 15 independent lines carrying either the P-E::GUS/P-355::3Δ orthe P-FL::GUS/P-35S::3Δ genes were developed. Of these, 7 lines carryingthe P-E::GUS/P-35S::3Δ genes, and 6 lines carrying theP-FL::GUS/P35S::3Δ genes exhibited abnormal phenotypes. In contrast, noabnormal phenotypes were observed in transgenic lines with reporter geneconstructs alone, or reporter genes plus the Rf2a gene. The abnormalT_(o) transgenic lines carrying P-E::GUS/P-35S::3Δ orP-FL::GUS/P-35S::3Δ genes were characterized by downward curving of themid-vein (FIGS. 6D,E,F), and the most severely affected plants werestunted (as shown in FIG. 6A). Two of the affected plants exhibitedabnormal floral development: these lines did not produce seeds

To confirm that the abnormal phenotypes were due to the transgene, weanalyzed the T₁, generation of the abnormal plant lines that gave seeds.Among all of the 11 lines tested, the abnormal phenotype was inheritedthrough the second generation. Line 1 of plants carryingP-FL::GUS/P-35S::3Δ showed an abnormal segregation pattern, while thesegregation of phenotype in other lines, for the most part, followedclassical, single locus segregation patterns. Abnormal plants grew muchmore slowly than the normal plants in T₁ progeny, and were characterizedby stunting of shoots and roots (as shown in FIGS. 6A, Q). The abnormalphenotype of T₁ plants that contained the P-E::GUS/P-35S::3Δ andP-FL::GUS/P-35S::3Δ genes can be characterized as either mild (as shownin FIG. 6B) or severe (as shown in FIG. 6F). Plants with the mildphenotype grew much more slowly than non-transgenic control plants butthey ultimately achieved normal height. These plants had wrinkled ordistorted leaves (as shown in FIGS. 6D, E) with yellow and green mosaicleaf color and exhibited reduced apical dominance and the appearance oflateral shoots (as shown in FIG. 6B, see arrows). Plants that exhibiteda severe phenotype were stunted with very short internodes, thick leaflamina, and an increased number of side shoots (as shown in FIG. 6F).The developmental problems in plants were severe and the plants did notproduce fertile flowers.

Expression of the 3Δ gene in the T₁ progeny was confirmed by northernblot analysis (data not shown). There was no correlation betweenseverity of the abnormal phenotype and the accumulation of mRNA derivedfrom the transgene.

GUS activity of transgenic lines with the P-E::GUS/P-35S::3Δ orP-FL::GUS/P-35S::3Δ genes were analyzed. The transgenic lines that didnot show an abnormal phenotype had the same levels of GUS activity asthe lines that contained only the reporter gene (i.e. P-E::GUS orP-FL::GUS) and there was no clear indication of gene activation orrepression. Some of the lines that exhibited abnormal morphology (i.e.,lines j, n, o of P-E::GUS/P35S::3Δ; lines e, h, k of PFL::GUS/P-35S::3Δ)had a similar level of GUS activity as transgenic lines that containedonly the reporter genes. No correlation was established between severityof plant phenotype and GUS activity. In some lines with severelyabnormal phenotypes, GUS activity increased with the age of the plants.These plants were physiologically different from normal plants and wesuggest that factors other than those related to the 3& mutant proteinmay affect expression of the RTBV promoter in these plants.

METHODS

Plasmid constructions. The complete RTBV promoter (nucleotides—731 to45) ligated with the uida coding sequence was released from plasmidpMB9089 (7) by digestion with Xbal and KpnI. The chimeric genecomprising the E fragment of the RTBV promoter (nucleotides—164 to +45)and the uidA coding sequence was released from plasmid pMB9089 (7) bydigestion with Hind III and Kpn I. Inserts were purified and ligatedinto the binary vector pGA482 through XBA I and Kpn I or Hind III andKpn I sites. The resultant plasmids are named pGA-FL::GUS and pGAE::GUS,respectively (as shown in FIG. 1B).

To construct plasmids for in vitro expression of Rf2a and the bZIP DNAbinding domain of Rf2a (referred to as 3Δ) (as shown in FIG. 1A) theplasmid pET-12a, which contains the cDNA encoding Rf2a, (11) was used.The Rt2a coding sequence was amplified by PCR from the starting plasmidby using primers 5′Rf2a (GCCGCCCATATGGAGAAGATGAACAGGGAGAAATCC) (SEQ IDNO: 3) and 3′Rf2a (CGCGGA TCCTCAGTTGCCGCTGCTTCCTGA) (SEQ ID NO: 4). Ndeland BamHI sites were introduced into the 5′and 3′primers, respectively(underlined). The coding sequence for 3Δ,comprising amino acids 108 to283 of Rf2a (11), was amplified by PCR from the Rf2a cDNA by usingprimers 5′ΔPΔQΔA-Rf2a (GCCGCCCATATGGAGAAGATGTCCGCCGCCGCCCA) (SEQ ID NO:5) and 3′Δ-Rf2a (CGCGGATCCTCAGTGTGGCATGCCACCGAA) (SEQ ID NO: 6). Asdescribed above, Nde I and Bam HI sites were introduced into theprimers. Pfu DNA polymerase (Stratagene) was used in all PCR reactions.The PCR products were digested with Nde I and Bam HI and inserted intopET28a (Novagen) restricted by the same enzymes. The new constructs werenamed pET-Rf2a and pET-3ΔThe sequence of inserts in each plasmid wasconfirmed by DNA sequencing.

Plant transformation vectors for co-expression of the reporter gene andthe effector proteins were constructed. The coding sequences of Rf2a and3Δ insert were released from pET-Rf2a and pET-3Δ with Nde I and Bam HI.The DNA fragments were then cloned into the plant expression vectorpMON999 (a gift from Monsanto Co.), which contains the CaMV 35S promoter(P35S) and Nos terminator, after the plasmid was restricted by Xba I(blunted with Klenow plus dNTPs) and Barn HI. The fusion genes namedP35S::Rf2a or P35S::3Δ were released by NotI and blunted with Klenow4polymerase plus dNTPs. The two fusion genes were then cloned into binaryvectors pGA-E::GUS or pGAFL::GUS through the blunted Clal site. In theco-expression vectors, the reporter gene and effector genes are in headto tail orientation. The final plasmids were namedpGA-E::GUS/P35S::Rf2a, pGA-FL::GUS/P-35S::Rf2a, PGAE::/GUS/P-35S::3Δ andpGA-FL::GUS/35S.3Δ (as shown in FIG. 1B).

Tobacco transformation. Gene constructs containing pGA482 derivedplasmids (as shown in FIG. 1B) were introduced into Agrobacteriumtumefaciens strain LBA4404 by electroporation and used forAgrobacteriurn-mediated transformation. Leaf discs from Nicotianatabacum cv. Xanthi NN were used for transformation following theprotocol described by Horsch et al (25). At least 14 independenttransgenic lines for each construct were produced and grown in agreenhouse. The transgenic plants were self-fertilized and T₁ seeds werecollected. For the analysis of the T₁ generation, seeds were germinatedon Murashige and Skoog (MS) medium (26) with or without kanamycin (100mg/l) selection and the seedlings were grown in a greenhouse.

Analysis of GUS activity. Histochemical analysis of B-glucuronidase(GUS) activity was performed essentially as described by Jefferson et al(27). Hand-cut fresh tissue sections of leaves and stems of primarytransformants T₀ or T₁, progeny were incubated at 37° C. for 4 to 12hours in reaction buffer containing 1 mM 5-bromo4-chloro-3-indolylglucuronide (X-gluc) (Research Organic), 100 mM sodium phosphate bufferpH 7, 2 mM potassium ferrocyanide and potassium ferricyanide, 0.1%Triton X-100 and 20% methanol. For analysis of young T₁ seedlings, wholeplatelets were collected about one week after germination and immersedin the buffer containing X-gluc. After vacuum infiltration, incubationwas carried out overnight at 37° C. Samples were cleared by severalwashes with 70% ethanol. Stained sections were immersed in 75% glyceroland visualized by optical microscopy.

Quantitative GUS analyses using the substrate 4,methylumebelliferone-β-S glucuronide (MUG) were performed as describedby Jefferson et al. (27).

ELISA. To detect Rf2a in extracts of transgenic tobacco leaves, sandwichELISAs were performed. 96-well microfilter plates (Nunc, MaxiSorp) werecoated overnight at 4° C. with Protein A at 1 ug/ml in PBS, 100 ul/well(Pierce). Plates were incubated for 1 hour at room temperature inblocking buffer [1×PBS containing 10% (v/v) Fetal Bovine Serum. (FBS)].Plates were incubated with the purified anti-Rf2a antibody (7) at 1ug/ml in 1×PBS plus 1% (v/v) FBS, for 1 hour at room temperature. Plantextracts in 50 mM Tris-HCl, pH 7.5, 0.1% v/v) Triton×100 were added andthe plates were incubated overnight at 4° C. Residual protein A bindingsites were blocked with protein A at 1 ug/ml in 1×PBS plus 1% (v/v) FBS.Plates were incubated again with the purified anti-Rf2a antibody, 1ug/ml in 1×PBS plus 1% (v/v) FBS, for 1 hour at room temperature, andthen with protein-A-HRP (Pierce) at 1 ug/ml in 1×PBS plus 1% (v/v) FBSand developed with TMB (KPL, Maryland, USA). Plates were read at 650 nmwith an absorbency plate reader. Between each incubation step, fivewashes with 1×PBS were performed. Purified Rf2a was used to create astandard curve to quantify the amount of Rf2a in each sample.

Production of Rf2a and pET-3Δ in E. coli, pET-Rf2a and pET-3Δ weretransformed into E. coli BL21 (DE3)/pLysE. Cells were grown at 37° C. toan OD₆₀₀ of 0.5-0.6, induced by 0.5 mMisopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h at 250C. The cellpellets were resuspended in 20 mM Tris-HCl pH 8, 500 mM NaCl, 0.1% NP40,1 mM PMSF, 1 mg/ml lysozyme, 10 ug/ml DNAase and 10 ug/ml RNase. Lysiswas completed using a French press. Under these conditions the targetproteins were soluble, and were dialyzed against Silvana and stored at−80° C.

Electrophoretic mobility shift assays (EMSAs). EMSAs were carried outessentially as described by Yin and Beachy (7). 500 ng of E. coliprotein extracts were incubated with 32_(P) labeled DNA probescomprising Box IIml that was constructed using annealed oligonucleotides(10). For competition EMSA, unlabeled oligonucleotides were added to thebinding reactions at 80 fold molar excess relative to the labeled probe(see FIG. 5).

REFERENCES

The following references are incorporated by reference.

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TABLE 1 Segregation of the abnormal phenotype in the R1 generationAbnormal Morphology/Total Morphology Construct Plant Lines in the R0Line Abnormal Normal E::GUS (+) 3Δ 7/15 i 7 3 k 16 4 l 5 5 m 5 0 n 7 3 o10 12 FL::GUS (+) 3Δ 6/15 e 7 3 h 7 3 k 14 7 l 2 8 n 8 1Predicted amino acid sequence of RF2a

MNREKSPIPG DGGDGLPPQA TRRAGPPAAA AAAEYDISRM PDFPTRNPGH RRAHSEILSLPEDLDLCAAG GGDGPSLSDE NDEELFSMFL DVEKLNSTCG ASSEAEAESS SAAAHGARPKHQHSLSMDES MSIKAEELVG ASPGTEGMSS AEAKKAVSAV KLAELALVDP KRAKRIWANRQSAARSKERK MRYIAELERK VQTLQTEATT LSAQLALLQR DTSGLTTENS ELKLRLQTMEQQVHLQDALN DTLKSEVQRL KVATGQMANG GGMMMNFGGM PHQFGGNQQN FQNNQAMQSMLAAHQLQQLQ LHPQAQQQQV LHPQHQQQQP LHPLQAQQLQ QAARDLKMKS PMGGQSQWGDGKSGSSGNPredicted amino acid sequence of RF2b (SEQ ID NO: 2)

MQEPKHTDPA AMRGAHHRRA RSEVAFRLPD DLDLGGGGAG AFDEIGSEDD LFSTFMDIEKISSGPAAAGG SDRDRAAETS SPPRPKHRHS SSVDGSGFFA AARKDAAASL AEVMEAKKAMTPEQLSDLAA IDPKRAKRIL ANRQSAARSK ERKARYITEL ERKVQTLQTE ATTLSAQLTLFQRDTTGLSA ENAELKIRLQ AMEQQAQLRD ALNDALKQEL ERLKLATGEM TNSNETYSMGLQHVPYNTPF FPLAQHNAAR QNGGTQLPPQ FQPPRPNVPN HMLSHPNGLQ DIMQQDPLGRLQGLDISKGP LVVKSESSSI SASESSSTF

1. A method of achieving constitutive expression of a gene of interestin plant cells, which comprises: (i) transforming the plant cells with afirst nucleic acid sequence that comprises the gene of interest operablylinked to a RTBV promoter, (ii) transforming the plant cells with asecond nucleic acid sequence conferring the constitutive expression of(SEQ ID NO: 1) the coding sequence for Rf2a, and (iii) growing the plantcells, wherein said constitutive expression of the gene of interest isachieved in the plant cells.
 2. The method of claim 1, wherein the plantcells are not rice cells.
 3. A plant transformation vector, capable oftransforming plant cells to achieve constitutive expression of a gene ofinterest, comprising (i) a RTBV promoter operably linked to the gene ofinterest and (ii) a constitutive promoter operably linked to a Rf2aencoding sequence.
 4. The vector of claim 3, wherein the constitutivepromoter is a CaMV 35S promoter.
 5. The vector of claim 3, wherein theplant cells are not rice cells.
 6. A set of plant transformationvectors, capable of transforming plant cells to achieve constitutiveexpression of a gene of interest, comprising (i) a first vectorcomprising a RTBV promoter operably linked to the gene of interest and(ii) a second vector comprising a constitutive promoter operably linkedto a Rf2a encoding sequence.
 7. The vector of claim 6, wherein theconstitutive promoter is a CaMV 35S promoter.
 8. The vector of claim 6,wherein the plant cells are not rice cells.